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Biomolecular Engineering 24 (2007) 119–124
Early stages of human plasma proteins adsorption probed
by Atomic Force Microscope
K. Mitsakakis, S. Lousinian, S. Logothetidis *
Aristotle University of Thessaloniki, Department of Physics, Solid State Physics Section, GR-54124 Thessaloniki, Greece
Abstract
Atomic Force Microscope (AFM) as a surface characterization technique has offered a great impulse in the advance of biocompatible materials.
In this study AFM was implemented for the investigation of the early stages of adsorption of two human plasma proteins on titanium and
hydrogenated carbon biocompatible thin films. The plasma proteins that were used were Human Serum Albumin and Fibrinogen, two of the most
important proteins in human plasma. The concentration of the protein solutions was the same as that in human plasma. As the examined samples
were soft, non-contact AFM mode was used to avoid their destruction. In order for the early stages of protein adsorption to be assessed, small
incubation times were applied. AFM measurements in liquid buffer were also carried out, allowing the observation of the protein behaviour in an
environment much closer to their native one. In addition, there was an assessment of the adsorption mechanism of the proteins on the above-
mentioned biomaterials.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Plasma proteins; Atomic Force Microscopy; Human Serum Albumin; Fibrinogen
1. Introduction
Atomic Force Microscopy (AFM) (Binnig et al., 1986) has
been implemented as a surface characterization technique for
the examination of biomolecules in various cases in biology-
biomaterial research (Hansma et al., 1997; Frederix et al., 2004;
Vansteenkiste et al., 1998). In the present work, it was involved
in the investigation of human plasma protein adsorption on
various biocompatible thin films. AFM offers the significant
advantage of probing in high detail the surface topography
qualitatively (by surface images) and quantitatively (by
mathematical quantities like surface roughness) due to its
nanometer-scale spatial resolution, both lateral and vertical
(Erlandsson et al., 1988; Hansma et al., 1988; Jandt, 2001).
AFM has proved to be very helpful for the determination and
verification of various morphological features and parameters,
like special molecular shapes and protein clusters, cluster size,
surface coverage, etc.
Protein adsorption, on the other hand, is a process that takes
place in effect on the interface of liquid solution and solid
substrate. In general, it is a quite multi-parametric procedure as
it is regulated by numerous factors like hydrophobicity, pH,
* Corresponding author. Tel.: +30 2310 998174; fax: +30 2310 998390.
E-mail address: [email protected] (S. Logothetidis).
1389-0344/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bioeng.2006.05.013
surface roughness, chemical composition, etc., and this
diversity of parameters makes it complex (Silva, 2002; You
and Lowe, 1996). However, it is of great importance, as a
process, when it comes to contact of biological matter, like
human plasma proteins which is of interest in this study, with a
biomaterial. Especially in haemocompatibility studies, the
adsorption of plasma proteins plays a key role, as it is the
proteins themselves that first come into contact with the
external biomaterial and promote or prevent the formation of
thrombus at the site of the ‘‘foreign’’, to plasma, biomaterial.
Human Serum Albumin (HSA) and Fibrinogen (Fib) are two of
the most important human plasma proteins. More particularly,
Fib takes part in blood coagulation, facilitates adhesion, and
aggregation of platelets, and is important in the processes of
both haemostasis and thrombosis, whereas HSA is believed to
act controversially to Fib, although its specific action is not yet
clear (Cacciafesta et al., 2000; Ortega-Vinuesa et al., 1998).
That is the reason why these two proteins were selected for the
purpose of our study.
Experiments have shown that amorphous hydrogenated
carbon (a-C:H) exhibits haemocompatible behaviour, and that
is why the main study was focused on a-C:H; Ti was
implemented as a reference material. Therefore, we were
motivated to use these materials as substrates for protein
adsorption (Logothetidis et al., 2005; YuU et al., 2000;
Vinnichenko et al., 2004; Logothetidis, 2002).
K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124120
2. Experimental
Titanium and a-C:H thin films were used for the study of protein adsorption.
More particularly, there were two types of a-C:H thin films, both deposited on c-
Si(1 0 0) wafer with RF reactive magnetron sputtering in a high vacuum
chamber with H2 (10%) as the reactive gas; one was deposited with biased
voltage (�40 V) and the other was deposited with no substrate bias (floating)
(Logothetidis, 2002; Gioti and Logothetidis, 2003).
The two proteins that were used in the experiments are HSA and Fib. HSA is
a heart-shape protein with approximate dimensions of 8 nm � 8 nm � 3 nm
(Sugio et al., 1999), while Fib is a more extended molecule with
length � width � height approximately 50 nm � 6 nm � 9 nm (Jandt, 2001;
Cacciafesta et al., 2000). Solutions of HSA and Fib in phosphate-buffered saline
(PBS, pH 7.4) were prepared and the incubation procedure was as follows; a-
C:H thin films were dipped into the protein solutions then the samples were
rinsed with deionized water and dried with mild N2 flow. The concentrations of
the solutions were 40 and 5 mg/ml for HSA and Fib, respectively—these are the
concentrations in human plasma as well. For both different samples, the
solutions were left for 5 and 10 min. Complementary measurements with
AFM were performed in liquid buffer as well.
Measurements were performed with SOLVER P47H Scanning Probe Micro-
scope (NT-MDT, NTI Instruments) in ambient and liquid environment. For
measurements in air, standard silicon cantilevers with nominal spring constant
11 N/m and resonance frequency 227 kHz were used for both cases. Because
protein samples are delicate and easy to be destroyed by the sharp AFM tip, Semi/
Non-Contact operation mode was employed. This way, the oscillatory motion of
the tip above the surface allows practically no contact with the sample and
therefore no danger to drag, deform, or scratch the latter exists. When it comes to
liquid measurements, contact operation mode was utilized. Cantilevers with
spring constant as soft as 0.1 N/m probed the surface morphology of the proteins
in their near-physiological environment. In either case, square images of the
samples were taken, with various sizes; 1 mm � 1 mm and 500 nm� 500 nm or
less when they would reveal interesting information. Quantities that were used for
the evaluation and comparison of the acquired data are peak to valley distance
(peak-to-peak), and root-mean-square roughness (Rrms).
3. Results and discussion
The haemocompatibility study of the a-C:H thin films used
in this work (through Spectroscopic Ellipsometry) has shown
that the film deposited under floating conditions presents much
Table 1
The film types, incubation times, and morphology parameters of surfaces,
including peak-to-peak distance and RMS roughness
Film type Incubation
time (min)
Peak-to-peak
(nm)
RMS roughness,
Rrms (nm)
a-C:H (biased) 0 5.160 0.566
HSA/a-C:H (biased) 5 13.980 1.255
HSA/a-C:H (biased) 10 6.695 0.842
Fib/a-C:H (biased) 5 19.020 2.363
Fib/a-C:H (biased) 10 6.630 0.910
a-C:H (floating) 0 15.840 2.131
HSA/a-C:H (floating) 5 24.900 2.758
HSA/a-C:H (floating) 10 16.200 2.365
Fib/a-C:H (floating) 5 21.840 2.776
Fib/a-C:H (floating) 10 20.526 2.576
Ti 0 10.560 0.994
HSA/Ti 5 13.140 1.022
HSA/Ti 10 10.560 1.048
Fib/Ti 5 16.260 1.849
Fib/Ti 10 12.000 1.278
HSA/a-C:H (floating) In liquid 14.040 1.846
Fib/a-C:H (floating) In liquid 17.940 2.286
better haemocompatible behaviour than the one deposited
under application of negative bias voltage (Logothetidis et al.,
2005). There has also been a study of protein adsorption
through AFM technique for longer incubation times (>10 min)
(Lousinian et al., 2007). This is the reason why the early stages
of protein adsorption is studied on these two samples, in this
work. The parameters that varied were: (1) different thin film
for protein adsorption; (2) different protein that was studied; (3)
different incubation times. Therefore, comparisons of results
will be made on three directions, each time keeping the rest of
two constant. In Table 1 and Fig. 1 details about peak to valley
distance and RMS roughness are presented, for incubation
times 0 min (bare substrate), 5, and 10 min, for HSA and Fib
proteins. This is why there will be several references to them
within this work.
From Table 1 and Fig. 1 it is noticed that the range of Rrms
values in the case of biased a-C:H is wider than in the case of
floating a-C:H. The relative changes in Rrms for Fib and HSA
from 0 to 5 min incubation times are 317.5 and 121.7%,
respectively, for the case of biased a-C:H. Equivalently, the
relative changes in Rrms for Fib and HSA from 0 to 5 min
incubation times in the case of floating a-C:H are only 30.3 and
29.4%, respectively. The large difference in relative roughness
values between biased and floating a-C:H thin films (for 5 min
incubation time) does not necessarily imply that there is more
protein material adsorbed on the former than the latter.
However, the fact that the surface roughness increases in both
cases, does mean that during the first 5 min there is definitely
some amount of proteins adsorbed on the surfaces.
By measuring and comparing the ‘‘bare’’ a-C:H substrates
through AFM images (Figs. 2a and 4a), it is clearly observed
that floating a-C:H substrate exhibits higher surface roughness
than the biased a-C:H, which is expected, since the sputtered
carbon atoms are distributed more evenly on Si substrate under
the influence of bias voltage.
Fig. 1. Comparative diagram of RMS roughness of examined plasma proteins
vs. incubation time for the two different types of a-C:H thin films and Ti thin
film.
K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124 121
Fig. 3. Topography image of: (a) Fib on a-C:H (biased) after 5 min incubation time (scan size 1 mm � 1 mm). Arrows indicate the molecular cluster features of
fibrinogen, (b) Fib on a-C:H (biased) after 5 min incubation time, with a 500 nm � 500 nm size focus on one of the fibrinogen molecular cluster—shape features
(Inset: the 2D equivalent image), and (c) Fib on a-C:H (biased) after 10 min incubation time (scan size 1 mm � 1 mm).
Fig. 2. 1 mm � 1 mm topography image of: (a) a-C:H (biased) as deposited, (b) HSA on a-C:H (biased) after 5 min incubation time (Inset: 500 nm � 500 nm size focus
on protein cluster), and (c) HSA on a-C:H (biased) after 10 min incubation time (Inset: 3D topography image, where the height differences are more clearly visible).
Fig. 2a shows the surface of biased a-C:H film without
proteins. It appears to have grain-like surface features, the size of
which is around 20–30 nm. Fig. 2b is the 5-min image of HSA on
biased a-C:H, and it is seen clearly that protein aggregates exist.
Typical dimensions are 100 nm � 180 nm and 11 nm height for
the bigger aggregate and 70 nm � 70 nm and 8 nm in height for
the small one, whereas the yellow circles denote regions with
other aggregates, with dimensions approximately 70–80 nm and
7–8 nm in height (it is worth noticing that in all the above-
mentioned cases, the proteins tend to ‘‘spread laterally’’, rather
than forming hills by accumulation of one on the other, since the
Fig. 4. 1 mm � 1 mm topography image of: (a) a-C:H (floating) as deposited, (b)
(floating) after 10 min incubation time.
height of the clusters is approximately once or twice the height of
one HSA molecule, while the lateral dimensions are larger).
Fig. 2c shows that in 10 min of incubation time, there are larger
protein clusters and the surface is partially covered. This is also
quantitatively verified by the decrease (even small) in the Rrms of
HSA, since aggregates dispersed on a relatively smooth surface
increase its roughness.
It was mentioned before that the Rrms of biased a-C:H thin
film is lower than that of floating a-C:H. That allows the
observation of molecular features of adsorbed proteins on
biased a-C:H. More specifically, in Fig. 3a the AFM reveals, in
HSA on a-C:H (floating) after 5 min incubation time, and (c) HSA on a-C:H
K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124122
Fig. 5. 1 mm � 1 mm topography image of: (a) Fib on a-C:H (floating) after 5 min incubation time and (b) Fib on a-C:H (floating) after 10 min incubation time.
fact, the packing of fibrinogen molecules. Because such a detail
is of great interest, images at smaller scan sizes (Fig. 3c) were
taken in order to focus on those regions. It was revealed, thus,
that the three-lobe structures that appear, are indeed, small
cluster of few molecules of fibrinogen – forming either
extended or V-shape conformations – that still retain the shape
of a single molecule (the size and morphology features fit those
mentioned in bibliography on TiO2 or mica (Jandt, 2001;
Cacciafesta et al., 2000; Marchant et al., 2002)). Such
‘‘protrusions’’ of the molecules cause the surface roughness
to increase, whereas in the case of 10 min incubation time
(Fig. 3b), Rrms is lower, due to the fact that more protein
material is deposited on biased a-C:H, ‘‘smoothening’’, thus, its
morphology (Fig. 1).
As far as the floating a-C:H thin film is concerned, the grain
sizes have maximum size, again, around 30–40 nm (Fig. 4a) (in
contrast with maximum 20–30 nm in biased a-C:H films) and
the percentage of big grains is greater than in the case of biased
a-C:H. These differences account for the 3.5� greater Rrms or
floating a-C:H thin films.
Comparing now the images of 5 and 10 min of incubation time
of HSA on floating a-C:H (Fig. 4b and c) one can note that both
the number and the size of the protein clusters are greater, which
means that more surface coverage is achieved. It cannot be
securely deduced, however, what percentage of surface has been
Fig. 6. 1 mm � 1 mm topography image of (a) Ti (as deposited), (b) HSA on Ti af
cluster), and (c) Fib on Ti after 5 min. Incubation time.
covered; only that in 10 min floating a-C:H is more covered,
relatively to the incubation time of 5 min. Decrease of Rrms
indicates gradual completion of surface coverage in these early
stages of protein adsorption (which may not be total till the
10 min, but certainly is more than in 5 min) during which both
protein–protein and surface–protein interactions are important.
From quantitative results from images of fibrinogen
solution of 5 min incubation on floating a-C:H (Fig. 5a), it
comes that Rrms for Fib- and for HSA-covered surface (both for
5 min and floating a-C:H film) is around the same value,
whereas the former is twice higher than the latter (2.36 nm
versus 1.26 nm) for the case of biased a-C:H film under the
same incubation conditions (Fig. 1). In addition, it is
remarkable that no special features are seen, as in the
equivalent case of biased a-C:H. One explanation is that the
surface roughness of the film is so high that does not allow any
shaped features to be ‘‘visible’’. On the other hand, another
possible case is that fibrinogen does not adsorb on floating a-
C:H in the same way that it does on biased a-C:H. However, this
speculation needs further investigation.
Although the main material of investigation was a-C:H, Ti
films on Si(1 0 0) were used as well as a substrate for protein
adsorption. From Table 1 it can be seen that Rrms values
for HSA on Ti are practically the same for both incubation
times. This ‘‘unchanging’’ behaviour could mean that since,
ter 5 min. incubation time (Inset: 200 nm � 200 nm focus on the HSA protein
K. Mitsakakis et al. / Biomolecular Engineering 24 (2007) 119–124 123
Fig. 7. 1 mm � 1 mm topography image in PBS liquid buffer of: (a) HSA on a-C:H (floating) in and (b) Fib on a-C:H (floating).
by the addition of a protein solution on the surface of Ti, hardly
any changes on its morphology take place, HSA adsorption on Ti
films is not favoured. On the contrary, Rrms for fibrinogen is
nearly doubled, revealing that changes on the surface of Ti took
place, i.e., Fib adsorption. After all, from Fig. 6b and c it can be
seen that in 5 min, only few HSA clusters exist (raising, thus,
negligibly the Ti substrate roughness), whereas for the same
time elapsed, several Fib aggregates – with size from 40 nm
and more, in contrast to the relatively small Ti grains of 30 nm
maximum size – are dispersed on Ti surface.
Finally, for qualitative reasons, measurements in liquid
were conducted. The processes of sample-to-tip approaching,
finding an appropriate region and scanning took several
minutes after the incubation of the solution drop on
the floating a-C:H surface, and due to that inherent limitation
in time that was imposed, the results are not indicative of
the early stage adsorption procedure (images were succeeded
to obtain in 30–35 min after incubation). Yet, they give a
qualitative view of the biomaterial surface when proteins are
in their near-physiological environment. What Fig. 7a and b
show, in fact, is that after several minutes of incubation time,
the protein–protein interactions prevail, since protein atoms
of the solution ‘‘meet’’ protein atoms on the surface as
they adsorb (instead of biomaterial atoms) and, thus, due to
the affinity of a protein molecule with the rest of its kind, the
surfaces show nearly the same morphology. However, Rrms
for Fib is somewhat greater than that of HSA, possibly due to
uneven settling of fibrinogen molecules on one another
and due to the fact that fibrinogen molecules are quite larger
than HSA.
4. Conclusions
From the present study, it is verified that protein adsorption
of HSA and fibrinogen is substrate dependent. This fact is
apparent both qualitatively (from images, by noting differences
in surface coverage, protein cluster sizes, protein morphology
features, etc.) and quantitatively (from variations in RMS
roughness). Fig. 1 indicates that Rrms exhibits an increase and
decrease at 5 and 10 min of incubation time, respectively,
nearly for all the examined cases. In addition, the Rrms values
for 10 min resemble those of bare floating a-C:H. These could
lead to the conclusion that at first, protein clusters form on
biomaterial surface, at distance from one an other, and then, as
protein material is added, they coalesce to form (at greater
incubation times possibly) a protein layer that fully covers the
surface, although the fact that both a-C:H and Ti have a grain-
like surface morphology (unlike mica or HOPG, which are
atomically smooth) makes it difficult to evaluate precisely the
degree of surface coverage by proteins.
It is obvious that the questions on protein adsorption cannot
be answered by a single technique, and require real-time and
kinetic experiments as well, but with this work it was proved
that AFM can contribute significantly to this direction. Further
steps on this work include verification of the above results with
contact angle measurements as well as imaging with Scanning
Near-Field Optical Microscopy. The former will assist the real-
time evaluation of the adsorption rate and the observation of the
profile of a protein solution drop, while the latter will exhibit
the competitive adsorption behaviour in the case incubation in
solution with both proteins.
Acknowledgement
One of us (K.M.) acknowledges financial support of Public
Benefit Foundation Alexander S. Onassis.
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